More than 60 million people have been infected with the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS), since the early 1980s. See Lucas, 2002, Lepr Rev. 73(1):64-71. HIV/AIDS is now the leading cause of death in sub-Saharan Africa, and is the fourth biggest killer worldwide. At the end of 2004, an estimated 39.4 million people were living with HIV globally, and still newly infected people with HIV is amounting to 4.9 million at the end of 2004 (source: UNAIDS).
Antiviral therapy targets different stages of the HIV life cycle and a variety of enzymes essential for HIV's replication and/or survival. Amongst the drugs that have so far been approved for AIDS therapy are nucleoside reverse transcriptase inhibitors (NRTI) such as AZT, ddI, ddC, d4T, 3TC, abacavir, non-nucleoside reverse transcriptase inhibitors (NNRTI) such as nevirapine, efavirenz, delavirdine, protease inhibitors such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir and lopinavir, entry inhibitors, etc.
In the absence of antiviral therapy, most HIV-1-infected individuals progress to AIDS and death. The median time between seroconversion and the development of AIDS is approximately 10 years (Easterbrook, 1999. J. Infect.). Rates of disease progression, however, are highly variable, ranging from rapid progression to AIDS within 1 year to long-term asymptomatic survival for over 15 years.
Several lines of evidence support an association between viral phenotype and rate of HIV-1 disease progression. Long-term non-progressors who harbor HIV-1 with mutations in nef have been described, and the viruses infecting those individuals have been characterized as less fit than wild-type viruses from individuals with progressive disease (Blaak et al. 1998. J. Infect. Dis.). Assays that measure the contribution of reverse transcriptase (RT) and protease (PR) to virus replication have been used to show that drug-resistant HIV-1 isolates have impaired replicative capacity. For instance, in Diallo et al. (Antimic. Agents and Chem., 2003) it is reported that a M184V substitution in HIV-1 reverse transcriptase, encoding high-level resistance to lamivudine (3TC), results in decreased HIV-1 replicative capacity, diminished RT processivity, and increased RT fidelity in biochemical assays.
In addition, diminished fitness of these isolates has been hypothesized to explain the clinical benefit of antiretroviral therapy in the setting of persistent virus replication. Further evidence for a link between virus replication rate and disease progression is suggested by the results of a study that showed that HIV-1 harbored by three long-term survivors had significantly less replicative fitness in growth competition experiments compared with HIV-1 harbored by three individuals with progressive disease (Quinones-Mateu, et al. 2000. J. Virol.). Campbell et al. (2003, J. Virol.) concluded that differences in HIV-1 replication rates among HIV-1 isolates are a major determinant of disease progression. As such, changes in the replication rate of a virus are of major clinical importance because they can affect the response of a patient to antiviral therapies, and are indicative of disease progression.
Fiebig Eberhard W. et al. in “Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection” AIDS (Hagerstown) vol. 17, no. 13, 2003, concludes that the quantitative analysis of preseroconversion replication rates of HIV is useful for projecting the yield and predictive value of assays targeting primary HIV infection.
WO04/003513 provides a method for determining the replication capacity of HIV. The method is based on an analysis of a panel of recombinant virus vectors created using site-directed mutagenesis containing one or more reverse transcriptase (RT) amino acid substitutions. The method basically detects whether the RT encoded by a HIV exhibits the presence or absence of a mutation associated with impaired replication capacity at amino acid position 98, 100, 101, 103, 106, 108, 179, 181, 188, 190, 225 or 236 of the amino acid sequence of said reverse transcriptase, wherein the presence of said mutation indicates that the HIV has an increased likelihood of having impaired replication capacity.
The method described in WO04/003513 thus relies on the knowledge of pre-existing data which associates specific RT mutations with a given replication capacity. Such method is thus not able to determine the replication rate of a diverse viral population encompassing other RT mutations, let alone protease mutations or any other enzymatic changes.
Campbell et al. (2003, J. Virol.) disclose a method for determining HIV-1 replication rates, said method encompassing the steps of culturing autologous virus isolates in phytohemagglutinin-treated peripheral blood mononuclear cells (PBMC), and determining the rate of p24 antigen production during its phase of exponential increase by fitting by linear regression, whereby the slope of the regression is the viral replication rate. Campbell et al. provide as well a method for determining the RT and PR replication capacity in a single cycle-based assay using recombinant virus that contain the RT and PR genes of each HIV-1 isolate. The replication capacity is the percentage of virus replication relative to the reference virus strain, NL4-3.
There is still an unresolved problem when determining the replication rate of viral stock of unknown titer. The titer of a viral population indicates the strength or potency of said viral population in infecting cells. The titer of a specific viral population can be defined as the highest dilution of said viral population giving a cytopathogenic effect (CPE) in 50% of inoculated cell cultures. Viral stocks which have a much too high or a much too low titer are usually difficult to measure because the indicator signals thereof fall out of the limits of detection of the analytical instrument used in said methodologies.
There is thus a need for a method for determining the replication rate of a viral population of unknown titer.
There is also a need for a method for determining the replication rate of a viral population which is not limited to specific mutant strains exhibiting specific RT mutations.
There is as well a need for a method for determining the replication rate of a viral population which is standardized, which can mimic an in vivo setting, is easy to use and easy to quantify in a precise manner. Importantly, there is the need of a superior method in terms of accuracy for determining the replication rate of a viral population.
It is an object of the invention to provide a method for determining the replication rate of a viral population with an unknown viral titer.
It is an object of the invention to provide a method for determining the replication rate of a viral population consisting of different viral types, strains, and quasispecies, and is further not limited to specific mutant strains.
It is an object of the invention to provide a method for determining the replication rate of a viral population which does not need to employ primers, probes, or any other analytical compounds designed and validated for each virus studied.
It is an object of the invention to provide a model for determining the replication rate of a viral population which can mimic in vivo conditions, is easy to use and easy to quantify in a precise manner.
It is an object of the invention to provide a method for determining the replication rate of a viral population which is accurate in estimating the replication rate.
It is an object of the invention to provide a method for determining the replication rate of a viral population which tests the complete full cycle of replication thereof.
It is an object of the invention to provide a method for determining the replication rate of a viral population in any chosen environment.